Hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content via increased formation or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. These often stem from altered P450 turnover elicited by drug-mediated P450 stabilization i.e. ethanol (EtOH), or enhanced drug-mediated P450 degradation (i.e. grapefruit juice). P450s such as CYP3A4, the major human liver/intestinal P450, and the EtOH-metabolizing CYP2E1 are degraded via ubiquitination by gp78 and CHIP E3 ubiquitin (Ub)-ligases and the 26S proteasome (UPD). We found that multisite protein phosphorylation considerably enhances gp78- and CHIP-mediated ubiquitination of P450 Lys-residues residing within negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) clusters. We hypothesized that P450 S/T-phosphorylation boosts the negative charge of these clusters enhancing its molecular E3-recognition, and regulating its UPD. We tested this hypothesis via state-of-the-art chemical-crosslinking/proteomic analyses, site-directed mutagenesis of the relevant CYP3A4 D/E/S/T clusters and each positively charged (K/R/H) gp78 patch and confirmed its validity. We propose such electrostatic interactions between D/E/S/T clusters and positively charged E3 patches are fundamental to E3 substrate recognition. Thus, our Aim 1 is to test this hypothesis by examining other hepatic P450s and/or cognate gp78- and CHIP-substrates with established crystal structures through similar experimental approaches. Because very few substrates are currently known, our Aim 2 is to employ SILAC coupled Ub-remnant analyses of gp78-/- and CHIP-/- and corresponding wild type mouse hepatocytes to identify additional hepatic E3-substrates that may be critically involved in pathophysiological relevant processes. CHIP-/- mice exhibit age-dependent hepatic oxidative damage, elevated lipid peroxidation and fat accumulation. We have shown that this is largely due to CYP2E1 stabilization, futile oxidative cycling, proteotoxic reactive O2-species, and activation of ASK1/JNK1 signaling pathways that are attenuated by CYP2E1 functional inhibition. By contrast, gp78-/- mice reportedly exhibit a very different phenotype. We hypothesize that gp78-/- livers fail to stabilize functional CYP2E1, as CHIP is fully capable of ubiquitinating it and diverting it into the alternative autophagic-lysosomal degradation (ALD) pathway, thereby effectively preventing its pathogenic accumulation. Although, both gp78 and CHIP E3s can independently ubiquitinate CYP2E1 required for its UPD, as an E4 gp78 also elongates Ub-chains via pro-UPD K48-Ub linkages. In its absence, CHIP effectively ubiquitinates CYP2E1 possibly via K63-Ub-linkages that route it into ALD. Our Aim 3 proposes to elucidate this pathophysiologically relevant CHIP-E3 versus gp78-E4 role in CYP2E1 degradation and consequent hepatic oxidative damage, lipid peroxidation and fat accumulation, clinical hallmarks of alcoholic liver disease, diabetes, obesity and non-alcoholic fatty liver disease (NAFLD).